Laporan isolasi dan pemeliharaan protoplast padi.
1990
Ambarwati D.A. | Somantri I.H.
A rice protoplast experiment was conducted in 1991/1992 at the Tissue Culture Laboratory, Bogor Research Institute for Food Crops. The main objective of the experiment was to isolate protoplasts from leaf mesophyll, 6 week-old callus derived from mature embryos and cell suspension culture of some rice varieties (Rojolele, Pandanwangi, Cisadane). Three enzyme combinations i.e. cellulase 1 percent - macerozyme 0,5 percent -pectolyase 0,1 percent (Enzyme I); cellulase 1 percent - pectolyase 0,2 percent (enzyme II) and cellulase 1 percent - macerozyme 0,5 percent (enzyme III) were used for cell wall digestion. Data collected from the experiment was the optimum time for releasing protoplast using the three enzyme combinations. Results indicated that enzyme II was more effective than the others. Most of the isolated protoplasts derived from leaf mesophyll and callus showed round shaped and no indication of bursting. During the isolation process using enzyme I, protoplasts were easier to burst although the optimum time of incubation wasn't much different compared to enzyme II especially for protoplasts derived from callus. Whereas using enzyme III, the isolation time took a longer time and the number of released protoplasts was low. Some aggregates of the cells were sometimes difficult to be digested and isolated. The best protoplast isolation process, regardless of the enzyme combinations, was obtained from Rojolele rice variety. We failed to obtain viable cells from cell suspension culture with either LS liquid medium supplemented with 2 mg 2,4-D/l; 1 g proline; 50 mg tryptophan nor N6 liquid medium enriched with 2 mg 2,4-D/l; 1 g proline; 10 g maltose. After 3 months of culturing, the cell suspensions were found unsuitable for protoplast isolation. Observations indicated that the suspensions were full of large aggregates, rooty, and also contaminated. It can be deduced that the success of protoplast isolation depended upon the type and duration of incubation, sources of protoplast and genotype factor.
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